Significant progress in plant biotechnology during the last two decades has opened the opportunities for commercial scale molecular farming in plants. The production of recombinant proteins in plants is an attractive alternative to more traditional systems based on bacteria and yeasts. It has an obvious advantage over the existing systems built on fermenters production due to its lower cost and the easiness of upscaling the production by simply increasing the harvesting of plant biomass.
In general, molecular farming in plants can be achieved by stable or transient expression of a recombinant protein of interest (Franken et al., 1997, Curr. Opin. Biotechnol., 8, 411-416; Fischer et al., 1999, Biotechnol. Appl. Biochem., Pt 2, 101-108; Herbers & Sonnewald, 1999, Curr. Opin. Biotechnol, 10 163-168). Stable transgenic plants can be used to produce vegetative tissues or seeds rich in recombinant protein. Vegetative tissue can be used directly for the processing while the seeds are more suitable for long-term storage. However, to reach high level of protein expression in plants is not an ordinary task, especially for the production of proteins compromising the plant growth. It requires the development of appropriately regulated expression systems, thus allowing to switch on the protein production at the right stage of plant development.
Existing technologies for controlling gene expression in plants are usually based on tissue-specific and inducible promoters and practically all of them suffer from a basal expression activity even when uninduced, i.e. they are “leaky”. Tissue-specific promoters (U.S. Pat. No. 5,955,361; WO09828431) present a powerful tool but their use is restricted to very specific areas of applications, e.g. for producing sterile plants (WO9839462) or expressing genes of interest in seeds (WO00068388; U.S. Pat. No. 5,608,152). Inducible promoters can be divided into two categories according to their induction conditions—those induced by abiotic factors (temperature, light, chemical substances) and those that can be induced by biotic factors, for example, pathogen or pest attack. Examples of the first category are heat-inducible (U.S. Pat. No. 5,187,287) and cold-inducible (U.S. Pat. No. 5,847,102) promoters, a copper-inducible system (Mett et al., 1993, Proc. Natl. Acad. Sci., 90, 4567-4571), steroid-inducible systems (Aoyama & Chua, 1997, Plant J., 11, 605-612; McNellis et al., 1998, Plant J., 14, 247-257; U.S. Pat. No. 6,063,985), an ethanol-inducible system (Caddick et al., 1997, Nature Biotech., 16, 177-180; WO09321334), and a tetracycline-inducible system (Weinmann et al., 1994, Plant J., 5, 559-569). One of the latest developments in the area of chemically inducible systems for plants is a chimaeric promoter that can be switched on by glucocorticoid dexamethasone and switched off by tetracycline (Bohner et al., 1999, Plant J., 19, 87-95). For a review on chemically inducible systems see: Zuo & Chua, (2000, Current Opin. Biotechnol, 11, 146-151). Other examples of inducible promoters are promoters which control the expression of patogenesis-related (PR) genes in plants. These promoters can be induced by treatment of the plant with salicylic acid, an important component of plant signaling pathways in response to pathogen attack, or other chemical compounds (benzo-1,2,3-thiadiazole or isonicotinic acid) which are capable of triggering PR gene expression (U.S. Pat. No. 5,942,662).
There are reports of controllable transgene expression systems using viral RNA/RNA polymerase provided by viral infection (for example, see U.S. Pat. Nos. 6,093,554; 5,919,705). In these systems, a recombinant plant DNA sequence includes the nucleotide sequences from the viral genome recognized by viral RNA/RNA polymerase. The effectiveness of these systems is limited because of the low ability of viral polymerases to provide functions in trans and their inability to control processes other than RNA amplification.
The systems described above are of significant interest as opportunities of obtaining desired patterns of transgene expression, but they do not allow tight control over the expression patterns, as the inducing agents (copper) or their analogs (brassinosteroids in case of steroid-controllable system) can be present in plant tissues at levels sufficient to cause residual expression. Additionally, the use of antibiotics and steroids as chemical inducers is not desirable for the large-scale applications. When using promoters of PR genes or viral RNA/RNA polymerases as control means for transgenes the requirements of tight control over transgene expression are also not fulfilled, as casual pathogen infection or stress can cause expression. The tissue or organ-specific promoters are restricted to very narrow areas of applications, since they confine expression to a specific organ or stage of plant development, but do not allow the transgene to be switched on at will.
One way of achieving high level of protein production is transient expression, where the transgene can be delivered and expressed at the desired stage of plant development, fully exploiting plant resources and allowing high yield of the desired product. The transient expression approach most suitable for medium to large-scale production include Agrobacterium-mediated (Kapila et al., 1996, Plant Sci., 122, 101-108) and plant viral vector-mediated systems (for review see: Porta & Lomonossoff, 1996, Mol. Biotechnol., 5, 209-221; Yusibov et al., 1999, Curr. Top. Microbiol. Immunol., 240, 81-94). Viral vector-based expression systems offer a significantly higher yield of transgene product compared to plant nuclear transgenes. For example, the level of transgenic protein can reach 5-10% of the total cellular plant protein content, when expressed from a viral vector (Kumagai et al., 2000, Gene, 245, 169-174; Shivprasad et al., 1999, Virology, 255, 312-323). RNA viruses are the most suitable as they offer a higher expression level compared to DNA viruses. There are several published patents which describe viral vectors suitable for systemic expression of transgenic material in plants (U.S. Pat. Nos. 5,316,931; 5,589,367; 5,866,785). In general, these vectors can express a foreign gene as a translational fusion with a viral protein (U.S. Pat. Nos. 5,491,076; 5,977,438), from an additional subgenomic promoter (U.S. Pat. Nos. 5,466,788; 5,670,353; 5,866,785), or from polycistronic viral RNA using IRES elements for independent protein translation (German Patent Application No. 10049587.7, PCT application PCT/EP01/11629). The first approach—translational fusion of a recombinant protein with a viral structural protein (Hamamoto et al., 1993, BioTechnology, 11, 930-932; Gopinath et al., 2000, Virology, 267, 159-173; JP6169789; U.S. Pat. No. 5,977,438) gives significant yield. However, the use of such an approach is limited, as the recombinant protein cannot be easily separated from the viral one. One of the versions of this approach employs the translational fusion via a peptide sequence recognized by a viral site-specific protease or via a catalytic peptide (Dolja et al., 1992, Proc. Natl. Acad. Sci. USA, 89, 10208-10212; Gopinath et al., 2000, Virology, 267, 159-173; U.S. Pat. Nos. 5,162,601; 5,766,885; 5,491,076).
Expression processes utilizing viral vectors built on heterologous subgenomic promoters provide the highest level of protein production to date (U.S. Pat. No. 5,316,931). The most serious disadvantage of such vectors and many others is their limited capacity with regard to the size of DNA to be amplified. Usually, stable constructs accommodate inserts of not more than one kb. In some areas of plant functional genomics this may not be such a serious limitation as G. della-Cioppa et al. (WO993651) described the use of TMV-based viral vectors to express plant cDNA libraries with the purpose of silencing endogenous genes. Two-component amplification systems which make use of helper viruses may offer a slightly better capacity (U.S. Pat. No. 5,889,191). However, for most applications, including production of proteins or low-molecular weight compounds in plants, these limitations cannot be remedied within existing processes. For example, in order to produce biodegradable plastics in plants, up to four recombinant genes must be expressed (Hanley et al., 2000, Trends Plant Sci., 5, 45-46) and at least seven bacterial genes are required for modulation of the mevalonate biosynthetic pathway in plants (Dewick, P., 1999, Nat. Prod. Rep., 16, 97-130).
A further serious concern with prior art virus-based plant expression systems is biological safety. On the one hand, high infectivity of the recombinant virus is highly desired in order to facilitate spread of the virus throughout the plant and to neighboring plants thereby increasing the yield of the desired gene product. On the other hand, such a high infectivity compromises containment of the recombinant material since spread to undesired plants may easily occur. Consequently, safer virus-based plant expression systems are highly desired.
Therefore, it is an object of the invention to provide a process of amplification or expression of a nucleic acid sequence of interest in a plant cell, which does not have the above-mentioned shortcomings and notably does not have the size limitation of the sequence of interest.
It is another object of the invention to provide a new process which allows amplification or expression of more than one nucleic acid sequences of interest in a plant cell.
Further it is an object of the invention to provide a process of amplification or expression of nucleic acid sequence(s) of interest in a plant cell, which is of improved ecological and biological safety.
Here, we describe a system that is devoid of the above limitations: it has no detectable limit on the size of DNA to be expressed, it allows expressing multiple genes in the same cell and plant and it possesses high built-in biosafety parameters.